The invention relates to an isolated oligopeptide, obtained from Entamoeba histolytica, which is useful in treating inflammatory conditions, such as rheumatoid arthritis and psoriasis. The isolated oligopeptide, which is identified herein as monocyte locomotion inhibitory factor (xe2x80x9cMLIFxe2x80x9d), is useful for suppressing leukocyte, macrophage and neutrophil activity which may be caused by an inflammatory event.
Entamoeba histolytica, in motile form, is a dynamic pleomorphic protozoon which is common in Mexico, Africa and Asia. E. histolytica is an invasive parasite having a simple cytoplastic structure. Infection by pathogenic E. histolytica may result in the invasion of several organs and tissues in humans. The most commonly affected organs are the colon and the liver. Less frequently, the parasite may invade the lungs, the brain, the skin and the genitalia.
E. histolytica is known to cause liver abscesses and other lesions in the human population. In amoebic liver lesions, a moderate inflammation occurs characterized by the presence of neutrophils, epithelioid cells and macrophages, with less abundant neutrophils, lymphocytes and plasma cells. It has been observed that although the early stages of parasitic invasion are characterized by acute inflammation in which even some eosinophilic leukocytes occur, the advanced stages are characterized by a scarcity of inflammation. E Moreover, livers with such hepatic abscesses have been found to regenerate perfectly without a trace of scarring following effective treatment with appropriate medicines. Sepulveda, B. et al., Immunology of Parasitic Disease, ppg. 170-191(1982).
The supernatant fluid of axenically grown E. histolytica has been shown to inhibit chemotaxis, chemokinesis and the random mobility of human mononuclear phagocytes. Human polymorphonuclear neutrophil phagocyte locomotion is apparently unaffected. It has been postulated that the inhibition of human mononuclear phagocytes by the entamoeba product contributes to the lack of inflammatory reaction observed in the advanced stages of invasive amoebiasis, and consequently and auspiciously, to the lack of scar tissue formation upon healing of amoebic lesions through regeneration. Kretschmer, R. R. et al., Parasite Immunology, 7, Pages 527-543 (1985). See also Rico, G. et al., Archives of Medical Research, 28(5), pages 235-236 (1997). This product may also constitute a defensive factor of the amoeba which is capable of reducing or blocking the inflammatory response of the host.
In basic terms, inflammation is a localized, protective response elicited by a foreign antigen, or by an injury or destruction of tissue. Inflammation occurs when tissues are injured by viruses, bacteria, trauma, chemicals, heat, cold, or any other harmful stimuli. In such instances, the classic weapons of the immune system (T cells, B cells, macrophages) interface with cells and soluble products which are mediators of inflammatory responses (neutrophils, eosinophils, basophils, macrophages, cytokines, kinin and coagulation systems, and the complement cascade).
A typical inflammatory response is characterized by (i) migration of leukocytes at the site of the injury or trauma; (ii) specific and nonspecific recognition of foreign antigens mediated by B and T lymphocytes, macrophages and the alternative complement pathway; (iii) amplification of the inflammatory response with the recruitment of specific and nonspecific effector cells by complement components, lymphokines and monokines, kinines, arachidonic acid metabolites, and mast cell/basophil products; and (iv) macrophage, neutrophil and lymphocyte participation in antigen destruction, with the ultimate removal of antigen particles or injured tissue by phagocytosis. Diseases associated with such inflammatory responses include rheumatoid arthritis, lupus and psoriasis. The rejection of allografts following organ transplantation also involves these inflammatory responses.
Although inflammation is an essential defense mechanism against infections, it is nevertheless appropriate to consider approaches to modulate or directly inhibit the inflammation if failure to do so would lead to severe and irreversible damage to organs and tissue, including non-functional scarring.
It will therefore be readily appreciated that a continuing need exists to develop improved treatments for inflammatory diseases, as well as treatments for conditions of moderate and extreme harmful inflammation.
It has now been discovered that oligopeptides obtained from the parasite Entamoeba histolytica have unique and selective anti-inflammatory characteristics which are useful for treating inflammatory conditions. The oligopeptides have been found to reduce the activity and mobility of leucocytes which are normally activated during an inflammation process in a subject. This oligopeptides are identified by the amino acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:2.
Accordingly, in one important aspect of the invention, compositions are provided comprising the oligopeptide set forth in SEQ ID NO:1.
In one embodiment of this aspect of the invention, the oligopeptide is a homodimer or a heterodimer. The homodimer comprises two oligopeptides as described in SEQ ID NO:1 linked together by, for instance, a cys-cys disulfide bond. A heterodimer comprises two oligopeptides, one of which is the oligopeptide as described in SEQ ID NO:1, which is, in effect, the active part of the heterodimer, and the other component is a different oligopeptide.
In another embodiment of this aspect, the oligopeptide is an analogue of SEQ ID NO:1. This oligopeptide contains a proline in place of the glutamine in the second position, and is set forth in SEQ ID NO:2.
According to another aspect of the invention, a method for treating a subject having a condition characterized by inflammation is provided. The method includes administering to the subject an amount of an oligopeptide, which includes the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, effective to reduce the inflammation in the subject. Inflammatory conditions which may be treated in this way include rheumatoid arthritis, lupus and psoriasis.
This aspect of the invention also includes suppression of allograft rejection following organ transplantation.
This aspect of the invention also includes the inhibition of leukocyte activity and mobility to prevent the occurrence of scarring in a patient. The oligopeptide is administered to the patient prior to the onset of the scarring process in the body or during healing.
In certain embodiments, the oligopeptide is administered to the subject as a treatment after the inflammatory condition has developed. In other embodiments, the compositions of this invention are formulated into pharmaceutical preparations to prevent the onset of the inflammatory condition, or, in another embodiment, as an anti-amoebic vaccine for immunization against amoebic invasion.
According to still another aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition includes an isolated oligopeptide which comprises the amino acid sequences set forth in SEQ ID NO:1 or SEQ ID NO:2, and a pharmaceutically acceptable carrier. The pharmaceutical composition also can include a compound which facilitates transport of the oligopeptide to the site of the inflammation. The pharmaceutical composition can be formulated into a product which can either be used for treatment or as a vaccine. The composition can include additives, excipients and adjuvants appropriate for treatment products or vaccines.
According to another aspect of the invention, nucleic acid sequences are provided. The nucleic acid sequences encode the oligopeptides identified in SEQ ID NO:1 and SEQ ID NO:2. Also included in the invention are vectors, such as expression vectors, which include the foregoing nucleic acid sequences. The nucleic acids can be used for medicinal applications, such as gene therapy applications.
In yet another aspect of the invention, oligopeptide-based compositions are provided for administration to a subject to prevent scarring which is the result of an inflammatory process mediated by leukocytes. Recruitment of fibroblasts, which are integral to the scarring process, is dependent on leukocyte macrophages. The activity and mobility of leukocyte macrophages is effectively blocked by the oligopeptide-based compositions of this invention.
The use of the foregoing compositions, isolated oligopeptides and isolated nucleic acids in the preparation of various medicaments is also provided.
These and other aspects of the invention are described in greater detail below.